Double strands of deoxyribonucleic acid (DNA) provide the essential information that defines the structure and function of most living organisms. Each strand of DNA is composed of a linear sequence of four nucleotide bases: adenine (A), thymine (T), guanine (G) and cytosine (C). The two strands of double-stranded DNA are complementary so that A pairs with T and pairs G with C. The sequence of bases in specific regions of the DNA are defined as genes since they determine the sequences of amino acids found in specific polypeptide chains of proteins. Other base sequences in the DNA participate in regulation of gene expression in that they play a role in defining when a gene is expressed so that protein is synthesized, and when a gene is not expressed. The differences in the sizes, shapes and other features among different species of organisms is largely explained by differences in the base sequences of their DNA. The DNA from individuals of the same species, however, can also vary in base sequence. Some of the variations in base sequence have little or no consequences, but others have dramatic effects. For example, a change or mutation in one base of the 2,000 or so bases in the sequence of the gene for .beta.-globin results in sickle cell anemia or other serious anemias in man. Single-base mutations or other changes such as deletions or insertion of bases in other genes cause diseases such as cystic fibrosis, Huntingdon's chorea, and osteogenesis imperfecta. For these and related reasons, detecting single-base mutations and other changes in DNA is of great importance for the treatment and diagnosis of diseases in humans, including the possibility of gene therapy. In addition, detection of mutations in genes is of great importance in a variety of other fields such as animal husbandry, development of new plant species and basic research. Ribonucleic acid (RNA) such as mRNA, is also composed of a linear sequence of nucleotide bases, adenine (A), uracil (U), guanine (G) and cytosine (C). Because RNA is derived from coding regions of DNA, mutations of a gene are also reflected in RNA.
Over the past several decades, a series of powerful techniques have been developed to precisely define the base sequences of fragments of DNA. The techniques involve chemical or enzymatic manipulation of the DNA followed by electrophoretic separation of the samples so that the exact sequence of bases in the DNA can be defined. However, defining the exact base sequences of a region of DNA or of a specific gene that may be as large as 200,000 base pairs continues to be a tedious and time-consuming undertaking for a number of reasons. For example, the number of manipulations is very large and for technical reasons, some DNA sequences are difficult to define. In addition, the amount of information that must be processed is very large and, therefore, errors are frequently made. Therefore, there is an important need for alternative procedures whereby short regions or fragments of DNA can be rapidly screened for the presence or absence of single-base mutations and other changes. More specifically, there is a need for methods whereby one can determine whether the base sequence in a test sample of DNA is or is not exactly the same as the base sequence in a second sample of standard DNA which may be the normal, unmutated or wild type DNA.
Analysis of DNA has been greatly facilitated by the development of the polymerase chain reaction (PCR) whereby a short region of approximately 1,000 bp of DNA can be amplified so that adequate amounts of the DNA are available for analysis. In addition, a number of techniques have been developed that can be used to detect single base mutations and other changes in the DNA amplified by the PCR or in DNA obtained by other procedures.
At least four general strategies have been pursued to develop such methods. One general strategy is to use enzymes that cleave DNA at sites in which a base is mismatched. In practice, the procedures involve preparation of heteroduplexes of DNA by first mixing a test DNA with a wild type DNA. The mixed sample is heated under conditions in which the double-stranded DNA will separate into single strands. Thereafter the mixture of single stranded DNA is cooled under conditions in which some single strands re-associate into double-stranded DNA having two completely complementary strands of base pairs (homoduplexes) and some single strands re-associate into double-stranded DNA in which most of the bases are complementary but one or more bases are not (heteroduplexes). An enzyme which recognizes mismatched DNA strands is then incubated with the mixture of DNAs so that it will cleave the heteroduplexes into two or more fragments that can be detected by separating the samples by electrophoresis. A second general strategy to detect mutations in DNA is based upon the principle that double-stranded DNA heteroduplexes will separate into two strands (dehybridize) under milder conditions than will double-stranded DNA homoduplexes. In practice, the principle is usually performed by electrophoresis of the DNA through gels which contain an increasing gradient of temperature or denaturants that promote the separation of double-stranded DNA into single-stranded DNA. Under appropriate conditions, heteroduplexes of DNA will partially separate into single-stranded molecules before homoduplexes of the same DNA. Therefore, the presence of a mutation can be detected by the slower migration in an electric field of heteroduplexes compared to homoduplexes.
A third general strategy is to use chemical methods either to modify unpaired bases or to modify and cleave unpaired bases in DNA heteroduplexes. In one variation of this strategy, heteroduplex DNA is cleaved by a chemical reagent at the site of a mismatch into two or more fragments. In another variation, one or both of the mismatched bases are modified by a chemical reagent that will preferentially modify bases that are not paired with a complementary base, but will not effectively modify bases that are paired with complementary bases in double-stranded DNA. The presence and usually the site of the chemically modified base can then be detected by a variety of techniques familiar to those skilled in the art.
A fourth general strategy is to detect mutations by separating or denaturing double-stranded DNA into single-stranded DNA and then comparing the electrophoretic mobility of the single strands of DNA to single strands of wild type DNA.
A number of variations of these four general strategies have been developed. Most of these strategies have been reviewed by Cotton, (1989) Biochemical Journal 263:1-10, and Ganguly and Prockop, (1990) Nucleic Acids Research 18:3933-3939. As indicated by Cotton in his review, however, each of the methods has limitations. For example, cleavage of mismatched bases in DNA heteroduplexes with the enzyme S1 nuclease was not sensitive enough to detect some single-base mismatches. Digestion of RNA-RNA heteroduplexes with the enzymes ribonuclease A or T1 was more sensitive but still detected only 60 to 70% of all possible single-base mismatches. Identification of single-base mutations by electrophoresis in denaturing gels was limited to the detection of mutations at low melting domains of DNA fragments and, therefore, required introduction of base sequences of Gs and Cs at one end of the DNA fragments to serve as clamps to delay the separation of the two strands. The technique also required careful optimalization of the conditions for electrophoresis for each DNA fragment. Chemical modification of DNA heteroduplexes with a water-soluble carbodiimide, a reagent that reacts with unpaired Gs and Ts, was shown to modify the electrophoretic behavior of DNA heteroduplexes containing six different single-base mismatches, but the effects on electrophoretic migration were often small and not readily detectable. In a modification of the carbodiimide procedure, antibodies specific for the carbodiimide-modified DNA were used to locate the site of single-base mismatches by immunoelectron microscopy of DNA heteroduplexes. The procedure, however, required time consuming electron microscopy. In still another variation of the carbodiimide technique, primer extension with DNA polymerase was used to locate the site of the mismatched base that was modified by carbodiimide. However, the procedure was time-consuming and required use of radioisotopes. Another chemical method involved modification of unpaired Cs in DNA heteroduplexes with hydroxylamine and unpaired Ts and Cs with osmium tetroxide. The DNA was then cleaved at the modified bases by treatment with piperidine and the resulting fragments were analyzed by gel electrophoresis to detect the site of the mismatch. The procedure was shown to detect mismatched bases in many sequence contexts but required preparation of radioactive probes to detect the cleaved fragments and as well as requiring two separate chemical reactions. At least one report (Bhattacharyya and Lilley, 1989 Journal of Molecular Biology, 209:583-593) indicated that the technique did not detect all single base mismatches in some sequence contexts. Detection of mutations by examining the electrophoretic mobility of single-stranded DNA is limited by the fact that the technique will detect mutations only in DNA fragments that are smaller than 300 bp and perhaps smaller than 200 bp. Also, the effect of a mutation on migration of the DNA strands is unpredictable and, therefore, the assays require careful analysis.
More recently, differential electrophoretic migration of homoduplexes and heteroduplexes of double-stranded DNA with single base mismatches has been described. For example, White et al. ( (1992) Genomics 12:301-306) report electrophoretic separation of homoduplexes and heteroduplexes of DNA fragments produced by the electrophoresis in polyacrylamide gel. White et al. produced a series of point mutations in a defined region of DNA from the equine infectious anemia virus. Each mutation was a single-base substitution. Eight of nine single-base mutations were detected by electrophoresis of heteroduplexes and homoduplexes of the DNA. It should be noted however, that the DNA sequence chosen by White et al. was a specialized base sequence that formed a hairpin-like structure in the DNA and, therefore, could be expected to be changed more dramatically by the presence of a single-base mismatch than would double-stranded DNA having more common tertiary structure. White et al. did not explore other sequence contexts. Most importantly, electrophoresis conditions for the separation of the homoduplexes and heteroduplexes was a standard buffer such as 25 mM Tris-borate and 1 mM EDTA together with 1% glycerol. In a few experiments mildly denaturing electrophoretic environment was created by adding 5% to 30% urea. White et al. found that 10% urea provided the greatest separation between homoduplexes and heteroduplexes. However, experiments have shown that most single-base mutations in heteroduplexes could not be detected using the method of White et al. Accordingly, there is a long felt need for a rapid, accurate method for detecting single base mutations in nucleic acid sequences. The present invention meets these and other needs which will become apparent through a reading of the following detailed description and accompanying claims.